We have previously identified four isoforms of the phosphatidylinositol 4-kinase enzymes (PI4Ks) that are expressed in Zebrafish and characterized their expression patterns during embryonic development. Although these enzymes catalyze the same biochemical reaction, their intracellular localization and presumably their regulation are different. Based on studies in yeast, these enzymes assume non-redundant functions. To identify developmental processes and signal transduction pathways in which specific phosphatidylinositol 4-kinases (PI4Ks) have pivotal roles, the expression of these enzymes were down-regulated by injection of morpholinos targeting the splicing of exons coding for the catalytic domains of the individual enzymes. [unreadable] In the first set of studies the role of PI4KIIIalpha was investigated. Down-regulation of PI4KIIIalpha resulted in multiple developmental abnormalities most severely affecting the hindbrain and the branchial arches. These changes were associated with highly increased apoptotic activity and were partially mimicked by treatment of the embryos with the PI 3-kinase inhibitor, LY294002. The most striking phenotype of PI4KIIIalpha down-regulation was the lack of pectoral fin development. Downstream targets of the FGF8 signaling pathway, such as MKP3 (a MAP kinase phosphatase) and Sef were strongly inhibited in the morphant embryos especially in the branchial arches and pectoral finbuds, whereas genes mediating hedgehog (Hh) signaling were only marginally affected. In HEK293T cells downregulation of PI4KIIIalpha by RNAi mediated gene silencing, inhibited constitutive Akt activation and potentiated the FGF- but not EGF-stimulated MAPK response. These data suggest that PI4KIIIalpha is critically important in supplying phosphoinositides for PI 3-kinases to activate anti-apoptotic pathways and that its down-regulation changes the balance between the MAPK and PI3K signaling pathways in FGF signaling causing a defect in pectoral fin development. Further studies are in progress to define the molecular details of how this PI4K isoform contributes to signaling from FGF receptors. [unreadable] [unreadable] Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 is the major phosphoinositide species in mammalian cells that has been associated with numerous molecular events critical for cellular signaling. PtdIns(4,5)P2 is hydrolyzed by phospholipase C enzymes to generate diacylglycerol and Ins(1,4,5)P3, two pivotal second messengers. PtdIns(4,5)P2 is also converted by Class I PI 3-kinases to PtdIns(3,4,5)P3, another important membrane-bound messenger molecule. PtdIns(4,5)P2 directly interacts with several ion channels, transporters, actin binding proteins, and regulates enzymes such as PLC and PLD. A number of molecules that are part of the receptor internalization machinery have also been shown to contain inositide binding domains but the exact lipid species that regulates them in the cell has not been firmly established. It is a major challenge to understand how a single type of molecule is able to regulate so many processes simultaneously and perhaps independently within the plasma membrane (PM). [unreadable] Last year we reported on the development of a new strategy to promptly regulate membrane PtdIns(4,5)P2 levels by a drug-inducible membrane targeting strategy based on the heterodimerization of the FRB domain of mTOR and FKBP12. In this approach the enzyme of interest, in this case a type-IV phosphoinositide 5-phosphatase (5-ptase,) was fused to the FKBP12 protein and upon addition of rapamycin (Rapa) the enzyme rapidly translocates to the membrane where its binding partner, the FRB domain, is targeted. We have shown that this manipulation rapidly eliminates PtdIns(4,5)P2 from the plasma membrane. [unreadable] This approach has been applied to study the PtdIns(4,5)P2 regulation of a number of ion channels and transporters. In collaboration with Dr. Rohacs, the phosphoinositide regulation of the TrpV1 and TrpM8 channels (the calcium channels responsible for hot and cold sensations, respectively) was compared. Intriguingly, while both of these channels are regulated by phosphoinositides, the regulation of TrpM8 is relatively simple, as it requires PtdIns(4,5)P2 for activity, and PtdIns4P cannot substitute for the former lipid. In contrast, TrpV1 channels are inhibited as well as stimulated by PtdIns(4,5)P2 depending on the dose of capsaicin, and PtdIns4P is also able to support its calcium channel activity. The molecular basis for this regulatory difference between these channels is currently under further investigation in the laboratory of Dr. Rohacs. In similar experiments we also showed that the Orai1 channel that supports capacitative calcium entry does not require PtdIns(4,5)P2 for its activity.[unreadable] In another collaboration with the group of Dr. Moolenaar in the Netherlands, the regulation of the GAP junction protein, connexin43, by PtdIns(4,5)P2 has been revealed. These studies showed that transport of small molecules from cell-to-cell via GAP junctions formed by the molecule, connexin43, requires PtdIns(4,5)P2 in the plasma membrane, and that decreasing the level of this lipid rapidly shuts down this form of intercellular communication. The Moolenaar group also showed that connexin43 regulation by PtdIns(4,5)P2 occurs via receptor activation of PLCbeta3, which associates with the connexin43 protein indirectly. [unreadable] Last year we also reported that PtdIns(4,5)P2 was required for the internalization of transferrin receptors. This observation was followed up in a collaboration with the group of Dr. De Camilli in which the role of PtdIns(4,5)P2 in the plasma membrane recruitment of several clathrin adaptor proteins was investigated. PtdIns(4,5)P2 depletion resulted in a rapid loss of clathrin puncta from the plasma membrane, which correlated with a massive dissociation of endocytic adaptors. The remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which are concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PtdIns(4,5)P2 in clathrin coat dynamics and Arp2/3-dependent actin regulation.